It is now clear that both CD4+ and CD8+ subsets contain CD45RA+ memory cells.. Over that period a vast literature has described the phenotype, location and behaviour of T cells derived f
Trang 1β 2 m = β 2 microglobulin; IFN- γ = interferon-γ; IL = interleukin; LFA-1 = lymphocyte function-associated antigen-1; NK = natural killer cells; PPD = purified protein derivative; RA = rheumatoid arthritis; SCID = severe combined immunodeficiency; SF = synovial fluid; TCR = T cell receptor; Th = T helper cells; TNF = tumour necrosis factor; TREC = T cell receptor excision circle.
Introduction
Despite many years of study, the aetiology of inflammatory
arthritis remains poorly understood A growing body of
data describing leukocyte differentiation, migration and
cellular interactions has put us in a promising position to
further dissect the molecular basis of inflammatory
arthritis A recent meeting brought together more than 60
researchers from across the UK at the Kennedy Institute of
Rheumatology, Imperial College, London The informal
atmosphere of the meeting encouraged the presentation
of recent results and novel ideas by 20 speakers covering
four themes
T cell activation and differentiation
Professor M Salmon (Birmingham University, UK) outlined
recent changes in the model of T cell differentiation in
which activation turns naive CD45RA+ T cells into CD45RO+primed/memory T cells, which divide periodically until they die It is now clear that both CD4+ and CD8+
subsets contain CD45RA+ memory cells Detailed study
of CD8 memory using MHC class I/viral peptide tetramers has defined several new models of CD8 differentiation according to the changing expression of numerous cell surface markers Memory CD45RA+cells are now widely accepted; their function, particularly proliferative potential,
is currently under debate Professor Salmon showed proliferation in CD8CD45RA+ memory cells, but only under stringent stimulation conditions; this may explain the poor responses reported for these cells These new concepts of differentiation have prompted re-examination
of T cells in arthritis Lymphocyte function-associated antigen-1 (LFA-1) and the chemokine receptor CCR7
Meeting report
Kennedy Institute of Rheumatology Division, Imperial College
London, 12–13 November 2003: Towards a molecular toolkit for
studying lymphocyte function in inflammatory arthritis
Jeff Faint1 and Frances Hall2
1 The University of Birmingham/MRC Centre for Immune Regulation, University of Birmingham, Birmingham, UK
2 University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, UK
Corresponding author: Jeff Faint (e-mail: j.m.faint@bham.ac.uk)
Received: 10 Feb 2004 Revisions requested: 11 Feb 2004 Revisions received: 18 Feb 2004 Accepted: 19 Feb 2004 Published: 8 Mar 2004
Arthritis Res Ther 2004, 6:55-59 (DOI 10.1186/ar1162)
© 2004 BioMed Central Ltd (Print ISSN 1478-6354; Online ISSN 1478-6362)
Abstract
T lymphocytes have been implicated in the pathogenesis of inflammatory arthritis for approximately
30 years Over that period a vast literature has described the phenotype, location and behaviour of
T cells derived from patients with inflammatory rheumatological disease The arthritiogenic roles of
MHC class I and class II molecules, which present antigen to T cells, have been hotly debated The T cell
has been variously conceived as a central or peripheral (or even incidental) component in the
arthritogenic response Rapid developments in genomics and use of biological therapeutic agents
coupled with recent insights in the field of T cell differentiation and immunoregulation together offer
novel methods of investigating the pathogenesis of chronic inflammatory disease A number of UK
researchers, with diverse interests within the field of synovitis, met recently at the Kennedy Institute of
Rheumatology Presentations on T cell memory, cytokines of homeostasis and inflammation,
unconventional behaviour of MHC molecules and immunoregulation in murine models, rheumatoid and
spondyloarthritis reflected the breadth of the discussion
Keywords: cytokines, HLA-B27, immunoregulation, migration, rheumatoid arthritis, spondyloarthritis
Trang 2discriminate the two CD45RA+ populations in healthy
subjects; naive cells are LFA-1lowCCR7high, memory cells
LFA-1highCCR7low [1] Dr J Faint (Birmingham University,
UK) has characterised CD8+CD45RA+ cells found in
rheumatoid synovial infiltrates Synovial CD8CD45RA+
cells are LFA-1high memory cells, containing Epstein–Barr
virus tetramer binding cells in seropositive subjects Some
synovial, but not blood, CD8CD45RA+ memory cells
expressed CCR7, which could be induced by culture in
rheumatoid synovial fluid (SF) CCR7 directs migration to
lymph nodes, with naive T cells migrating through high
endothelial venules, and maturing tissue dendritic cells to
afferent lymphatics These data suggest that tissue
infiltrating T cells might operate a similar mechanism to
return to draining lymph nodes
T cell differentiation in arthritis was also examined by Dr F
Ponchel (Leeds University, UK), using differential
expres-sion of CD45 isoforms and T cell receptor exciexpres-sion circle
(TREC) analysis [2] TRECs are not replicated during
division and provide an indication of the replicative history
of cell populations Patients with rheumatoid arthritis (RA)
had reduced frequencies of naive and ‘conventional’
memory cells compared with healthy donors, yet expressed
additional populations not evident in controls This might
result from lymphopoenia, which is a feature common to
many diseases Reduced bone marrow stromal cell
production of interleukin (IL)-7 in rheumatoid patients
leads to a lack of circulating cytokine, which was restored
in some patients by therapy with anti-tumour necrosis
factor-α (anti-TNF-α) antibodies
In addition to the alterations in subset frequencies, T cells
in rheumatoid patients are hyporesponsive to stimulation
through the T-cell receptor (TCR) Dr A Cope (Kennedy
Institute, Imperial College, London, UK) demonstrated that
TCR triggering leads to transient internalisation and
subsequent re-expression of TCR/CD3 Chronically
stimulated cells, particularly in the presence of TNF-α,
show sustained low-level expression of the ζ signalling
chain of the CD3 complex, impairing signal transduction in
these cells [3] TCRζdimcells express many markers typical
of highly differentiated, senescent effector cells, and
respond poorly to stimulation by CD3/CD28 The
rheumatoid synovium is highly enriched in TCRζdim cells,
which might explain their hyporesponsiveness, while also
suggesting that effector responses of these cells are
relatively independent of antigen signals Tissue-infiltrating
cells in arthritis seem to be chronically stimulated, yet it is
unclear how they compare with cells during an evolving
immune response Professor A Akbar (University College
London, UK) has modelled a cutaneous inflammatory
response to purified protein derivative (PPD), showing the
T cell infiltrate to be oligoclonal and extensively
proliferating; 80% of clones were maintained between
days 7 and 19 of the response T cell proliferation is
affected by telomere shortening (repeating sequences of DNA found at the chromosome ends) with each cell division, eventually leading to replicative senescence; the enzyme telomerase can also lengthen telomeres Shortened telomeres were seen from day 14, yet little telomerase
activity was evident in vivo Cells stimulated in vitro
upregulated telomerase This was inhibited by fluid obtained from inflammatory blisters and might explain the limitation of expansion of specific T cells at sites of inflammation Synovial infiltrating T cells have been extensively studied, yet they are far outnumbered by neutrophils Professor R Moots (Liverpool University, UK) has been dissecting the interactions between these cells Joint neutrophils are long lived and produce many inflammatory mediators Neutrophils from healthy subjects express MHC class II after culture in a cytokine cocktail, whereas rheumatoid joint neutrophils spontaneously express MHC class II after overnight culture [4] These neutrophils can present
superantigen to T cells in vitro, suggesting a possible
antigen-presenting function for neutrophils; however, this
activity has yet to be demonstrated in vivo.
Lymphocyte recognition of HLA-B27
The association of ankylosing spondylitis with HLA-B27 is among the strongest described for an HLA locus, giving
an odds ratio of about 161 HLA-B27 also exhibits associations with other spondyloarthropathies, including reactive arthritis, psoriatic spondyloarthritis and spondylo-arthritis associated with inflammatory bowel disease
Dr P Bowness (Weatherall Institute of Molecular Medicine, Oxford, UK) discussed the unusual biology of HLA-B27 molecules and the implications of this for recognition by T cells and natural killer (NK) cells Classically the heavy chain of MHC class I molecules folds
in association with β2microglobulin (β2m) and a nonamer peptide This monomer presents the peptide to TCRs on CD8+T cells Indeed, the HLA-B27 monomer is capable
of presenting several peptides derived from bacterial triggers of reactive arthritis HLA-B27 is unusually (although not uniquely) able to form heavy-chain homodimers, via a disulphide bridge at Cys 67 [5] Modelling this complex suggests that the α1 helix unfolds and that the peptide-binding groove can accommodate a peptide much longer than a nonamer Expression of HLA-B27 heavy chain in cells deficient in TAP (transporter associated with antigen processing) results in homodimer formation, demonstrated
by binding of homodimer-specific monoclonal antibody Mice transgenic for HLA-B27, but deficient for β2m, express large quantities of surface homodimer HLA-B27+
β2m+mice express the conventional monomeric molecule but low levels of homodimer are also detectable HLA-B27 homodimer tetramers have revealed that about 5% of peripheral CD8+ T cells are capable of recognising the HLA-B27 in this conformation Of great interest is the
Trang 3observation that homodimer tetramers also bind to B cells,
monocytes and NK cells Further work is under way to
characterise the profile of receptors that recognise the
homodimer, and the relationship of homodimer formation
to the development and activity of spondyloarthritis
Conventionally, HLA-B27/peptide is recognised by CD8+
T cells The hypothesis that some CD4+ T cells might
recognise HLA-B27 arose from observations in
spondylo-arthritic HLA-B27-transgenic mice [6] Despite the apparent
importance of HLA-B27 in the pathogenesis, CD8+T cells
were shown not to be essential for the development of
arthritis; indeed, disease was transferable with CD4+T cells
Professor JSH Gaston (University of Cambridge School of
Clinical Medicine, UK) discussed the characterisation of a
variety of unconventional T cell clones from patients with
spondyloarthritis Several patterns of specificity in these
CD4+clones have emerged: first, proliferation in response
to HLA-B27, either empty monomers or, possibly,
homo-dimers; second, proliferation in response to an
HLA-B27-derived peptide presented by HLA-Cw1; and third,
proliferation in response to an HLA-B27-derived peptide
presented by alloantigens, including HLA-B51 and HLA-A2
Dr H Bodmer (Edward Jenner Institute for Vaccine Research,
Newbury, UK) continued the discussion on the nature of
HLA-B27-restricted CD4+ T cells A double transgenic
mouse model expressing GRb (TCR specific for HLA-B27
presenting influenza nucleoprotein 383–391) and HLA-B27/
β2m revealed the presence of HLA-B27-restricted CD4+
as well as CD8+ T cells The CD4+ cell required the
presence of MHC class II to be selected, presumably by
dual α-chain expression [7] However, the selected CD4+
T cells responded to HLA-B27/NP(383–391) with similar
sensitivity to the CD8+T cells In vitro, both Th1 and Th2
differentiation of HLA-B27-restricted CD4+ T cells could
be achieved The expression of MHC class II molecules is
generally restricted to professional antigen-presenting
cells, so as to regulate the initiation of responses by CD4+
T helper cells Recognition of MHC class I molecules by
CD4+T cells could therefore have important implications
for the initiation of autoimmunity Professor Gaston also
discussed the isolation of unconventional CD8+ T cells
from patients with spondyloarthropathy These autoreactive
CD8+T cell lines/clones produce IL-4 but not interferon-γ
(IFN-γ) They constitutively express the αβ TCR, CD69,
CD25 and CD30 and express CD40L on activation; they
are perforin-negative and express only low levels of
granzyme These clones are restricted by autologous
MHC class I molecules and are dependent on an
unidentified (ubiquitous?) peptide The possibility that they
are CD8+T regulatory cells is under investigation
Immunoregulation
Immunoregulation is an integral part of normal innate and
adaptive immune responses Recently, the literature has
burgeoned with the reports of the regulatory properties of CD4+T cells which constitutively express CD25 It is clear that regulation does not reside exclusively within this group but has also been attributed to subsets of CD8+
T cells (see above), dendritic cells, NK cells, neutrophils and eosinophils, as well as to the plethora of soluble regulatory molecules A tangential but germane observation is that pathogenic microbes have developed
an astonishing capacity for host immunointerference Many multicellular parasites exemplify the delicate balance of immunomodulation without global immunosuppression; this permits chronic infection of the host, often over decades
Dr L Taams (Kings College, London, UK) presented comparisons of the phenotype and function of CD4+
CD25+T cells in healthy controls and in both peripheral
blood and SF of patients with RA In vitro, the
CD4+CD25+T cells from RA and control peripheral blood were equally potent at inhibiting the proliferation of CD4+CD25− T cells The CD4+CD25+ T cells derived from RA SF were more potent suppressors but, curiously, the CD4+CD25−T cells from SF were more resistant to proliferative suppression Peripheral blood CD4+CD25+
cells modified cytokine release in an antigen-specific stimulation of CD4+CD25− cells TNF-α production was reduced in CD4+CD25− cells from peripheral blood, whereas IFN-γ and IL-10 were both decreased in CD25−
cells from SF Continuing the theme of regulatory T cells in
RA, Michael Ehrenstein (Centre for Rheumatology Research, London, UK) discussed the change in function
of CD4+CD25+T cells in the peripheral blood of patients with RA before and after TNF-α blocking therapy In comparison with healthy controls, patients with RA demonstrated a defect in the ability of their CD4+CD25+
T cells to inhibit the production of TNF-α by monocytes This defect was unchanged by treatment with methotrexate but was restored in patients who were successfully treated with TNF-α blockade
Dr L Wedderburn (Institute of Child Health, London, UK) advanced the hypothesis that immune regulation contributes
to the mild phenotype of children with oligoarticular juvenile idiopathic arthritis compared with patients with extended oligoarthritis The CD25+ and particularly CD25hiT cells in the SF of patients with juvenile idiopathic arthritis exhibited a regulatory phenotype (GITR+, CTLA-4+, [FoxP3 mRNA]high) The regulatory activity of this subset was also supported by the demonstration that depletion of
SF CD25+cells releases the proliferation of CD25−cells
T cells in SF from children with oligoarthritis expressed an approximately ten-fold higher CD25 expression than T cells from patients with extended oligoarthritis, which is of particular interest because the most potent regulatory activity has been shown to reside in the CD4+CD25hi
subset Children with mild disease also had higher overall numbers of CD4+CD25+ cells in the SF Although
Trang 4suppressive potency per cell seemed comparable in cells
from patients with mild and severe disease, these
observations are consistent with the hypothesis that the
CD4+CD25+ regulatory subset is less effective in the
children who develop extended oligoarticular arthritis
Frances Hall (University of Cambridge School of Clinical
Medicine, UK) presented data from a murine model of
inflammatory arthritis that suggested a role for regulatory
T cells in the repression of arthritis and dermatitis Adult
DBA/1 males were thymectomised and then treated with
CD25-depleting antibody, which depleted both
CD4+CD25med and CD4+CD25hi cells Although a low
level of inflammatory arthritis is usually evident in elderly
DBA/1 males, the CD25-depleted mice developed more
severe arthritis Histologically, this was associated with
pannus formation and erosions In addition, about 60% of
the CD25-depleted mice also developed an ulcerating
rash, characterised by a dense neutrophilic infiltrate The
nature of the emerging inflammatory disease and the
influence of the genetic background of the mice are
currently under investigation
Dr L Pazmany (Clinical Sciences Centre, Liverpool, UK)
broadened the immunoregulatory discussion by considering
the role of NK cells NK cells are present at autoimmune
sites of pathology, including the rheumatoid joint [8] In the
murine model of extrinsic allergic encephalitis, depletion of
NK cells resulted in an exacerbation of disease For other
models of autoimmune disease, results of NK depletion
have been variable The effect of addition of autologous
NK cells to a PPD-pulsed culture of T cells and
autolo-gous antigen-presenting cells was investigated, using
proliferation as a read-out The effect of freshly isolated
NK cells was dependent on the donor However, NK cells
incubated with IL-12 reproducibly inhibited T cell
proliferation, even at NK:T ratios of 1:10 The significance
of these observations for the susceptibility of individuals to
RA and for the severity of disease is being investigated
Professor I McInnes (University of Glasgow, UK)
demonstrated how the immunoregulatory prowess of
multicellular parasites might inform our search for the
optimal disease-modifying agent The filarial protein ES62
promotes a Th2 response in the BALB/c mouse It seems
to bind to TLR4 and promotes the development of type 2
dendritic cells, thereby decreasing IL-12 and TNF-α
production by macrophages In the murine
collagen-induced arthritis model, serial subcutaneous ES62
administration decreases the severity, although not the
incidence, of disease [9] This is associated with a
decrease in the proliferation and production of IL-6, IFN-γ
and TNF-α by draining lymph node cells from bovine
collagen type II immunised mice This effect of ES62 is
mirrored by a decrease in the production of TNF-α and IL-6
in primary synovial membrane cultures Because parasitic
products such as ES62 might be well tolerated for decades, without global immunosuppression, they seem a promising therapeutic strategy for Th1-dominant inflammatory diseases, such as RA
Homing and effector function
Tissue-specific ‘address codes’ have been defined for skin and gut homing lymphocytes by using the pattern of chemokine receptors and adhesion molecules that they express Isolating specific microvascular vessels has proved difficult and has prevented the definition of a synovial tissue code Professor C Pitzalis (Guy’s Hospital, London, UK) outlined a novel approach to this problem by using phage display libraries constructed from blood vessels formed in human synovial tissue transplanted into SCID (severe combined immunodeficiency) mice [10] Initial results show promise in isolating joint-specific homing markers He also showed that the rheumatoid joint has a similar spatial organisation of lymphocytes and chemo-kines to that seen in lymph nodes Lymph node develop-ment requires careful and coordinated interactions between different cell types; these results indicate that similar inter-actions might occur in the joint The theme of cell–cell interactions was continued by Professor F Brennan (Kennedy Institute, Imperial College, London, UK) T cells prolong spontaneous TNF-α release from cultured rheuma-toid synovial membrane cells through cell contact with
macrophages Using an in vitro model, T cells activated by
TCR crosslinking induced monocytes to produce TNF-α and IL-10, whereas T cells stimulated with a cytokine cocktail (analogous to bystander activation) induced predominantly TNF-α production By using pharmaco-logical inhibitors it now seems that these distinct responses arise through the recruitment of different signal transduction pathways in responder macrophages Thus, phosphoinositide 3-kinase inhibitors reduced cytokine production induced by TCR-stimulated T cells, yet NFκB inhibitors enhanced the response The opposite responses
to inhibitors were seen if bystander T cells were used Importantly, rheumatoid synovial T cells behave like bystander activated cells, providing further clues to the mechanisms of T cell effector responses in the joint CD3− CD56+NK cells present in bystander activated populations also induced TNF-α from synovial membrane cells in a contact-dependent manner Joint-infiltrating CD56+ cells were shown by Professor M Callan (Imperial College, London, UK) to enhance TNF-α production by monocytes About 10% of NK cells from peripheral blood express higher levels of CD56 than the majority of cells, yet CD56bright cells were the predominant population infil-trating the joint [11] Attempts to differentiate CD56intinto CD56brightcells have failed, indicating that they represent two separate lineages CD56brightNK cells express low levels of perforin, mount poor cytotoxic responses and produce many proinflammatory cytokines on stimulation These activities might further exacerbate responses in inflammatory sites
Trang 5Professor J Isaacs (Newcastle University, UK) concluded
with some words of caution about our ability to develop
novel antibody therapies, which have generally proved less
effective in humans than in the mouse This might be due
to targeting the wrong molecules or the wrong epitopes of
those molecules, or to incorrect doses and duration of
therapy Tolerance might take time to develop, and
concomitant therapy might actually hinder its development
A further complication is that some therapies might initially
exacerbate symptoms before providing benefits This might
partly account for the clinical improvement seen in some
patients in antibody trials after treatment was stopped
owing to disease flares The challenge for the future will lie
in recognising what are side effects of treatment and what
are potentially dangerous turns in disease
Conclusions
The overwhelming message from this meeting was the
appreciation of not only the diversity of cell types present
in the inflamed joint but also the diversity of their
interactions No cell type seems to be solely responsible
for the maintenance of inflammation; rather, it is the
interactions between the multiple cell types present This
presents a number of potential targets for future therapies,
yet suggests that an effective cure will require multiple
interventions targeting multiple pathways
Competing interests
None declared
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Correspondence
Jeff Faint, Rheumatology Unit, Division of Immunity and Infection, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Tel: +44 121 415 8690; fax: +44 121 414 3599; e-mail: j.m.faint@bham.ac.uk